Production of Crystalline Short Chain Amylose

ABSTRACT

A process for producing a starch comprises treating a feed starch that comprises amylopectin with glucanotransferase to produce a chain-extended starch, and treating the chain-extended starch with a debranching enzyme to produce a starch product that comprises amylose fragments. At least about 38% by weight of the amylose fragments have a degree of polymerization (DP) of at least about 35.

This application claims priority from U.S. provisional patentapplication Ser. No. 60/715,832, filed on Sep. 9, 2005, which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

Starch comprises two polysaccharides: amylose and amylopectin. Amyloseis a generally linear polymer that comprises glucose units connected byalpha 1-4 glycosidic linkages. Amylopectin is a branched polymer inwhich many of the glucose units are connected by alpha 1-4 glycosidiclinkages, but some are connected by alpha 1-6 glycosidic linkages.

Alpha-amylase is an enzyme that is present in the human body and whichhydrolyzes alpha 1-4 linkages in starch, thus leading to digestion ofthe starch. In certain situations it is desirable to produce starch thatresists hydrolysis by alpha-amylase, for example to decrease the caloriccontent of the starch, or to increase its dietary fiber content.However, attempts to produce such starch in the past have suffered fromone or more problems, such as high cost.

Amylase-resistant starch is usually produced from high-amylose starch,which is often expensive. There is a need for improved processes forproducing starch with a high content of amylose that is suitable forproduction of alpha-amylase resistant starch.

SUMMARY OF THE INVENTION

One embodiment of the invention is a process for producing a starch. Theprocess comprises treating a feed starch that comprises amylopectin withglucanotransferase to produce a chain-extended starch, and treating thechain-extended starch with a debranching enzyme to produce a starchproduct that comprises amylose fragments. At least about 38% by weightof the amylose fragments have a degree of polymerization (DP) of atleast about 35. The process can optionally further include recoveringthe amylose fragments. As another option, the process can includemembrane filtering a solution or dispersion of the starch product toincrease the concentration of amylose fragments that have a degree ofpolymerization (DP) of at least about 35.

Another embodiment of the present invention is a starch product producedby the above-described process. In some embodiments of the invention, atleast about 40% by weight of the amylose fragments have a degree ofpolymerization (DP) of at least about 35. If the process used to makethe starch product includes membrane filtration, then in someembodiments at least about 50% by weight of the amylose fragments have adegree of polymerization (DP) of at least about 35. In some instancesthe starch product has a peak melting temperature of greater than about105° C. Amylose in the starch can be crystallized to increase itsresistance to alpha-amylase.

Another embodiment of the invention is a food product that contains theabove-described starch.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-45 are described in the following pages.

DESCRIPTION OF SPECIFIC EMBODIMENTS

One embodiment of the present invention is a process of producing starchhaving a relatively high content of amylose. This process includestreating a feed starch that comprises amylopectin withglucanotransferase to extend at least some of the starch chains, andtreating the chain-extended starch with a debranching enzyme to produceamylose fragments. These amylose fragments can then be crystallized toproduce a resistant starch product.

Ordinary dent corn starch can be debranched enzymatically to give shortchain amylose fragments, but since the amylopectin component of thestarch is usually composed of relatively short branched chains, theproduct contains too few of the longer chain lengths that are needed forenzyme resistance. Debranched dent corn starch that has not beenmodified with a glucanotransferase typically contains less than 35% ofthe DP35 and higher chain lengths (i.e., starch molecules having adegree of polymerization of at least 35) and therefore does not have thethermal stability needed for a resistant starch. In addition, thedebranched dent starch contains a fraction of long chain lengths fromamylose as well as short chains from amylopectin. This combination ofheterogeneous chain lengths is not optimal for crystallization andamylase resistance.

The feed starch used in the present process can come from a variety ofsources, including dent corn, waxy corn, high amylose ae genetic corn(ae is the name of a genetic mutation commonly known by corn breedersand is short for “amylose extender”), potato, tapioca, rice, pea, wheat,waxy wheat, as well as purified amylose from these starches, andalpha-1,4 glucans produced according to patent application WO 00/14249,which is incorporated herein by reference, and combinations of two ormore of these starch sources. Chemically modified starches, such ashydroxypropyl starches, starch adipates, acetylated starches, andphosphorylated starches, can also be used in the present invention. Forexample, suitable chemically modified starches include, but are notlimited to, crosslinked starches, acetylated and organically esterifiedstarches, hydroxyethylated and hydroxypropylated starches,phosphorylated and inorganically esterified starches, cationic, anionic,nonionic, and zwitterionic starches, and succinate and substitutedsuccinate derivatives of starch. Such modifications are known in theart, for example in Modified Starches: Properties and Uses, Ed.Wurzburg, CRC Press, Inc., Florida (1986). Other suitable modificationsand methods are disclosed in U.S. Pat. Nos. 4,626,288, 2,613,206 and2,661,349, which are incorporated herein by reference.

If the feed starch is a waxy starch, it can be at least partiallydebranched by treatment with a debranching enzyme prior to treatmentwith glucanotransferase. Suitable debranching enzymes for this purposeinclude pullulanase and isoamylase. This provides a source of fragmentsthat will be transferred by the glucanotransferase to the amylopectinnon-reducing ends, resulting in longer branched chains.

4-α-glucanotransferase [2.4.1.25] is an enzyme that catalyzes thetransfer of a segment of a 1,4-alpha-D-glucan to a new position in anacceptor, which can be glucose or another 1,4-alpha-D-glucan.Glucanotransferase will catalyze the transfer of a maltosyl moiety to amaltotriose acceptor, releasing glucose. The glucose released can beused as a measurement of enzyme activity.

A suitable assay for determining glucanotransferase activity is asfollows. In this assay, maltotriose is used as both substrate andacceptor molecule. Glucose is released in this reaction and can bemeasured after a modified version of the common glucoseoxidase/peroxidase assay. (Werner, W. et al (1970) Z. Analyt. Chem.252:224.) GOD-Perid solution can be obtained from a Glucose Release Kitfrom WAKO, or can be prepared with 65 mM sodium phosphate, pH 7including 0.4 g/l glucose oxidase (Sigma G6125 or G7773), 0.013 g/l HRP(Sigma P8125), and 0.65 g/l ABTS (Calbiochem #194430). A 0.04 N NaOHsolution is also used. The substrate solution is 1% maltotriose (0.1 gmaltotriose in 10 ml of 50 mM phosphate buffer at pH 6.0).

Standard curve:

Glucose solution: weight out 0.1806 g glucose into 500 ml MQ H₂O.

Dilutions for standard curve:

Concentration μL glucose solution μL MQ water 0.01 μmol 5 495 0.05 μmol25 475  0.1 μmol 50 450 0.25 μmol 125 375  0.5 μmol 250 250

120 μl of the substrate solution is preincubated at a selectedtemperature, e.g. 60° C., for 10 minutes. 20 μl of enzyme solution areadded to the substrate solution and the reaction mixture is incubated at60° for 10 minutes. The reaction is stopped by the addition of 20 μl of0.04N NaOH. 20 μl is then transferred to a 96 well microtiter plate and230 μl GOD-Perid solution is added. After 30 minutes at roomtemperature, the absorbance is measured at 420 nm. The enzyme activityis calculated relative to the standard curve of glucose in the range of0-0.5 μmol glucose. One unit (U) of activity is defined as the amount ofenzyme that liberates 1 μmol glucose/minute.

Treatment of the feed starch with glucanotransferase produces extensionsof the chains on the amylopectin molecules. This treatment can beperformed, for example, in aqueous solution or suspension at atemperature of about 70-100° C. and a pH of about 5.0-6.0. As a result,the DP35 and higher content of the end product increases to over 38%, orin some cases to over 40%, and the chain lengths are much more uniform,which is indicated by a polydispersity of 2-4, compared to about 8 fordebranched dent corn starch. In some embodiments of the invention, thedosage of glucanotransferase can be about 1-15 ml per 100 gram ofstarch, preferably about 5-12 ml/100 g. The glucanotransferase can becontacted with the starch in a single dose, or split into multipledoses. In one embodiment of the invention, the total dosage is splitinto three portions which are provided at separate times (for example,three separate doses of 2.5 ml/100 g each), with at least one hourbetween each. In some embodiments, the reaction temperature can be fromabout 75-85° C., and the reaction time can be less than about 8 hours,preferably less than about 6 hours.

Optionally, an additional starch-based material can be added to thechain-extended starch prior to debranching. For example, a maltodextrincan be added.

The resulting chain-extended starch can then be treated with adebranching enzyme, such as isoamylase or pullulanase, for example at atemperature of about 30-60° C. and a pH of about 4.0-5.0 to produceamylose fragments having desirable lengths. In certain embodiments ofthe invention, the dosage of isoamylase is about 1-10 mg per g ofstarch, preferably about 1-5 mg/g.

The DP35 and higher content can be enriched to over 50% by fractionationby microfiltration at an elevated temperature, such as about 60-120° C.,more typically about 60-90° C., and even more typically 70-85° C. Thedebranched, glucanotransferase-treated, starch product aftermicrofiltration can have a peak melting temperature greater than about105° C., and can contain at least about 80% by weight resistant starchafter heating in water to about 98° C.

Optionally, the product starch can be heat treated in aqueous solutionor suspension at a temperature of at least about 90° C., or in someembodiments at least about 98° C. This heat-moisture treatment canincrease the total dietary fiber (TDF) content of the starch in someinstances. For example, the heat moisture treatment can increase the TDFof the starch from about 15-35% to about 75-80% in some embodiments ofthe invention.

In one embodiment of the process, the feed starch is slurried in waterat 15% solids and the pH is adjusted to 5.5 with dilute NaOH. The slurryis placed in an autoclave and heated to 140° C. for 30 minutes. Aftercooling to 85° C. and adjusting the pH to 5.5, glucanotransferase isadded and allowed to react for 24 hours. The enzyme is deactivated byreducing the pH to below 3.0. The starch is redispersed by heating to140° C. for one hour and then cooled to 45° C., and the pH is adjustedto 4.5. Isoamylase is added and allowed to react for 18-24 hours. Themixture is heated to 85° C. for one hour to deactivate the enzyme. Ifnecessary, the product can be treated again with isoamylase by repeatingthe 140° C. heating and enzyme treatment at 45° C. and pH 4.5. Theproduct can then be fractionated to increase the content of longer chaincomponents. This can be carried out, for example, by microfiltration ofthe crystallized debranched product at a temperature of at least about80° C. using a ceramic membrane with a pore size of about 0.45 microns.After collecting 1.5 to 2.5 volumes of permeate relative to the volumeof the starting slurry, while maintaining the volume of the retentate byaddition of deionized water, the product is isolated by concentratingand spray drying or by centrifuging and oven drying the retentate.

The product produced by the process contains a high percentage ofamylose which is suitable for making starch that is resistant toalpha-amylase. The resistant starch can be added to a number of foodproducts to reduce their glycemic index, and increase dietary fiber andprobiotic effect in the colon.

Starch produced by this process can be used as a bulking agent or floursubstitute in foods, such as reduced calorie baked goods. The starch isalso useful for dietary fiber fortification in foods. Specific examplesof foods in which the starch can be used include bread, cakes, cookies,crackers, extruded snacks, soups, frozen desserts, fried foods, pastaproducts, potato products, rice products, corn products, wheat products,dairy products, nutritional bars, food for diabetics, and beverages.

The starch product, at least in some embodiments, is thermally stable inwater at a temperature of at least about 90° C., or in some cases atleast about 100° C., allowing it to be used in food products that willbe processed at high temperature and moisture conditions.

Certain embodiments of the invention are described in the followingexamples.

EXAMPLE 1

170 g of common corn starch (Minstar 2030) were slurried with 830 mlcity water in a 3000 ml glass beaker. The pH was adjusted to 5.5 withNaOH/HCl and the suspension carefully heated to 65-70° C. under constantstirring to form a thick gel. A lid was placed on the glass beaker whichwas then transferred to an autoclave. When the steam pressure hadreached 40 psi (140° C.) the conditions were maintained for 30 minutes,after which the autoclave was allowed to cool.

The cooked starch was transferred to a stirred glass reactor and theconditions adjusted to 85° C., pH 5.5. 1.07 ml T. thermophilusglucanotransferase, corresponding to 10 μg enzyme protein /g DS, wereadded and the reaction allowed to continue for 24 hours.

The reaction was stopped by lowering the pH to below 3.0.

EXAMPLE 2

In order to generate suitable donor molecules for theglucanotransferase, the following experiment was carried out. 175 g ofwaxy corn starch (Cerestar 04201) were slurried with 830 ml city waterin a 3000 ml glass beaker. The pH was adjusted to 5.5 with NaOH/HCl andthe suspension carefully heated to 65-70° C. under constant stirring toform a thick gel. A lid was placed on the glass beaker which was thentransferred to an autoclave. When the steam pressure had reached 40 psi(140° C.) the conditions were maintained for 30 minutes, after which theautoclave was allowed to cool.

The cooked starch was then partially debranched after it had beentransferred to a stirred glass reactor. The temperature was adjusted to55° C., pH 4.3, and 0.0872 g Pseudomonas amyloderamosa isoamylase(350,000 IA units/g, from Hayashibara), was added, corresponding to 200Isoamylase units/g DS. The reaction was then allowed to continue for 3hours.

After partial debranching, the temperature was raised to 85° C. and thepH adjusted to 5.5 with NaOH. 1.10 ml T. thermophilusglucanotransferase, corresponding to 10 μg enzyme protein /g DS, wereadded and the reaction allowed to continue for 24 hours.

The reaction was stopped by lowering the pH to below 3.0.

EXAMPLE 3

In order to test if the degree of modification of starch byglucanotransferase played a key role, 170 g of common corn starch(Minstar 2030) were slurried with 830 ml city water in a 3000 ml glassbeaker. The pH was adjusted to 5.5 with NaOH/HCl and the suspensioncarefully heated to 65-70° C. under constant stirring to form a thickgel. A lid was placed on the glass beaker which was then transferred toan autoclave. When the steam pressure had reached 40 psi (140° C.), theconditions were maintained for 30 minutes, after which the autoclave wasallowed to cool.

The cooked starch was transferred to a stirred glass reactor and theconditions adjusted to 85° C., pH 5.5. 1.10 ml T. thermophilusglucanotransferase, corresponding to 10 μg enzyme protein /g DS, wereadded.

After 2 hours a further addition of 1.10 ml T. thermophilusglucanotransferase was made and the reaction allowed to continue for 27hours.

A further addition of 1.10 ml T. thermophilus glucanotransferase wasthen made and the reaction allowed to continue overnight.

The reaction was stopped by lowering the pH to below 3.0.

EXAMPLE 4

A sample of the glucanotransferase treated dent starch from Example 1was received as a frozen slurry. After saving a 50 g sample of thethawed slurry, the remaining slurry (495.3 g) was diluted with 200 g ofdeionized water and pH adjusted to 6.5 with 5% NaOH. The slurry wasplaced in a stirred high pressure reactor. After purging with nitrogen,the reactor was heated to 140° C. for 1 hour, and then cooled to 105° C.The product was removed from the reactor by purging through a valveconnected to a dip-tube into a 3-neck round bottom flask. The flask wasplaced in a 45° C. water bath and the pH was adjusted to 4.5 by addingdilute HCl. When the temperature of the solution reached 45° C., 18 mg(300 units/gram of starch) of Hayashibara isoamylase was added. Thesolution was allowed to stir overnight. The enzyme was deactivated byheating to 85° C. for 1 hour.

EXAMPLE 5

A sample of glucanotransferase-treated, partially de-branched waxystarch from Example 2 was received as a frozen slurry. After saving a 50g sample of the thawed slurry, the remaining slurry (469.0 g) wasdiluted with 200 g of deionized water and pH adjusted to 6.5 with 5%NaOH. The slurry was placed in a stirred high pressure reactor. Afterpurging with nitrogen, the reactor was heated to 150° C. for 1 hour, andthen cooled to 105° C. The product was removed from the reactor bypurging through a valve connected to a dip-tube into a 3-neck roundbottom flask. The flask was placed in a 45° C. water bath and the pH wasadjusted to 4.5 by adding dilute HCl. When the temperature of thesolution reached 47.7° C., 18 mg of Hayashibara isoamylase was added.The solution was allowed to stir at 45° C. overnight. The enzyme wasdeactivated by raising the pH to 6.3 with 5% NaOH and heating to 85° C.for 1 hour.

EXAMPLE 6

A sample of glucanotransferase-treated dent starch from Example 3 wasreceived as a frozen slurry. After saving a 50 g sample of the thawedslurry, the remaining slurry (473.0 g) was diluted with 500 g ofdeionized water and pH adjusted to 6.5 with 5% NaOH. The slurry wasplaced in a stirred high pressure reactor. After purging with nitrogen,the reactor was heated to 140° C. for 1 hour, and then cooled to 95° C.The product was removed from the reactor by purging through a valveconnected to a dip-tube into a 3-neck round bottom flask. The flask wasplaced in a 45° C. water bath and the pH was adjusted to 4.5 by adding 2drops of acetic acid and a few drops of 5% NaOH. When the temperature ofthe solution reached 45° C., 40 mg (300 units/gram of starch) ofHayashibara isoamylase was added. The solution was allowed to stirovernight. After adjusting the pH to 6.0, the sample was heated to 95°C. in a water bath and stirred for 1 hour. The flask was then placed ina 45° C. water bath, pH adjusted to 4.5 with dilute HCl and 30 mg ofHayashibara isoamylase was added when the temperature of the solutionreached 45° C. After stirring overnight, the pH was adjusted to 6.0 andheated to 85° C. for 1 hour.

Molecular distribution data showed that this sample was not completelydebranched, indicated by the presence of a significant amount of >16,000Dalton material. The sample was pH adjusted to 6.5 with 5% NaOH andheated as described above to 140° C. for 1 hour in a high pressurestirred reactor. The sample was removed from the reactor and placed in aflask in a 55° C. water bath and pH adjusted to 4.5. After the solutionreached 55° C., 79 mg of Hayashibara isoamylase was added. Afterstirring at 55° C. overnight, analysis of this suspension showed thatdebranching was completed.

EXAMPLE 7

Results of gel permeation chromatography (GPC) analysis of debranchedstarches are shown in Table 1.

TABLE 1 Analysis of Debranched Glucanotransferase-Treated Starches DP DPDP DP DP DP DP Example Description 1-6 7-12 13-24 25-36 37-60 60-100100+ Mw Mn 2/5 Part. 5.8 13.9 28.0 22.1 18.9 9.1 2.2 n/a n/a debranchedwaxy + GT 1/4 Dent + GT 2.8 10.5 24.9 22.2 22.4 12.8 4.5 n/a n/a 3/6Dent + 3X 2.5 9.5 23.6 26.0 22.7 13.4 2.3 6200 3683 GT “DP” means degreeof polymerization. “Mw” means weight-average molecular weight. “Mn”means number average molecular weight.

EXAMPLE 8

Microfiltration was carried out in a system comprising a reservoir witha heating jacket connected to a recirculation pump and a housingcontaining a Millipore 0.45 micron ceramic membrane. The jacket washeated with a circulating oil bath and the membrane housing was heatedwith an electric heating tape. The membrane housing was generallymaintained at 10-15° C. higher than the reservoir temperature to preventcrystallization of debranched material in the membrane.

The debranched glucanotransferase-treated dent starch suspension fromExample 6 (1056.9 g at about 5% solids) was diluted with 297 g ofdeionized water and heated in the microfiltration reservoir withrecirculation to 80° C. and held for 1 hour before starting to drawpermeate from the membrane housing. As permeate was collected an equalvolume of deionized water was added to the reservoir. After 3360 gramsof permeate were collected, the retentate (1236 g) was withdrawn fromthe reservoir and allowed to cool in a beaker placed in a refrigerator.The retentate contained 34.1 g of dry solids and the permeate contained9.0 g of dry solids. The retentate was isolated by dilution of theslurry with 3A ethanol, filtering and drying.

The molecular weight of the debranched glucanotransferase-treated starchand the retentate and permeate fractions from microfiltration wereanalyzed by GPC. The retentate was tested for resistant starch (% RS).Resistant Starch as defined by Englyst (Eur. J Clinical Nut. 1992), 46,(Suppl. 2), S33-S50) is a measure of the amount of starch that isresistant to hydrolysis by porcine pancreatin alpha-amylase at 37° C.after two hours treatment. The result is given as a percent of theinitial dry starch weight.

The results are shown in Table 2.

TABLE 2 Microfiltration Fractionation of DebranchedGlucanotransferase-Treated Dent Starch (80° C.) Permeate Yield DP DP DPDP Sample vol. wt. % 37-60 60-100 100+ Mw Mn 37+ % RS Starting sample —— 25.2 13.7 0.8 5762 3708 39.7 — retentate 2.5/1 73.5 35.7 15.5 0.3 65395083 51.45 87.2 permeate 2.5/1 26.5 13.9 1.4 0.0 3883 3022 15.24 — InTable 2, “2.5/1” indicates that the sample was washed and that 2.5liters of permeate were collected per liter of starting sample.

In Table 2, “2.5/1” indicates that the sample was washed and that 2.5liters of permeate were collected per liter of starting sample.

EXAMPLE 9

GT enzyme treatment of dent starch:

Dent Starch Pearl-C (15%) was jet-cooked (285-290° F.), the pH wasadjusted to 5.7, 4-α-glucanotransferase (GT) [2.4.1.25] was added (10ml/100 g starch), and reacted at 80° C. for 4 hr. The starch slurry washeated to 140° C. for 1 hr, the slurry was incubated at 55° C., pH 4.5,isoamylase was added (1 mg /g starch) and the slurry was incubated for24 hr. The starch slurry was cooled to room temperature, and then storedat 4° C.

Debranching GT treated starch in DMSO solution:

Debranching of GT treated starch in an experiment in which STAR-DRI® 10maltodextrin (Tate and Lyle, Decatur, Ill.) was conducted in DMSOsolution. Dry starch (35 mg) was dissolved in 1 ml aqueous DMSO(DMSO:water=9:1 v/v) or wet samples (269 mg, 13% DS in GT treatedsamples) were dissolved in 0.9 ml pure DMSO. The starch solution washeated in boiling water bath with stirring for 3 hr. The starch solutionwas then cooled to 39° C., and 3.5 ml warm sodium acetate buffer (39°C., 50 mM) was added. 100 μl isoamylase [10 mg/ml isoamylase (1,280,000U/g solid) in 0.1 N NaOAc buffer, pH 4.5] was added to the starchsolution. The starch and isoamylase mixture was incubated in a waterbath at 39° C. for 2 hr. The starch and isoamylase mixture was heated inboiling water for 20 min, and then cooled down to 39° C. 100 μlisoamylase was added and the mixture was incubated for 16 hr. Afterdebranching, the starch solution was heated in boiling water bath for 20min, and cooled down to warm temperature. A 2 mL aliquot of the mixturewas diluted with 2 mL of pure DMSO. The DMSO mixtures (about 5 mgstarch/ml) were heated in a boiling water bath for 20 min, allowed tocool to warm temperature. Dowex MR-3 resin (0.5 g) was added to thestarch solution and shaken for 1 min to remove NaOAc. The starchsolution was filtered through a 0.45 μm pore size Millipore filterattached to a 3 ml syringe. The filtered samples were injected into theHPLC with SEC or GPC column.

FIG. 1 shows the total dietary fiber content (TDF) of different portionsof GT converted dent starch. If microfiltration was not used, theprecipitated converted starch was filtered using filter paper, and driedin the oven (50° C.). The TDF value was 16.8% before heat-moisturetreatment, and 80% after heat-moisture treatment. When themicrofiltration was used, the retentate had a TDF of 33.84% beforeheat-moisture treatment and 77.6% after heat-moisture treatment. Thepermeate had little solids precipitated in 4° C., so the TDF was notanalyzed. By drying everything in the retentate and permeate using adrying bowl in an oven (100° C.), the estimated solids was 71.4% forretentate and 28.6% for permeate.

FIG. 2 shows chromatograms of GT treated resistant starch that wasdebranched using 1 mg isoamylase/g starch in the reactor for 24 hr, andfurther debranched by analytical debranching. The microfiltrationretentate had a peak DP of about 35, while the microfiltration permeatehad a peak DP of about 14. The filter paper retentate had a peak DP ofabout 30.

FIG. 3 shows chromatograms of GT treated resistant starch that has beendebranched using 1 mg isoamylase/g starch for 24 hr in the process, andGT treated resistant starch further debranched by analyticaldebranching. FIG. 3 shows that GT converted starch was almost completelydebranched in the reactor using 1 mg isoamylase/g starch for 24 hr.

FIGS. 4 and 5 show percentages of different DP ranges of three portionsof GT converted starches. Microfiltration retentate had about 38% DP37-100, 59% DP 25-100, 10% DP 1-12, and 34% DP 1-24. Microfiltrationpermeate had about 12% DP 37-100, 26% DP 25-100, 40% DP 1-12, and 74% DP1-24. Filter paper retentate had about 33% DP 37-100, 52% DP 25-100, 14%DP 1-12, and 39% DP 1-24.

TABLE 3 Percentage of Different DP Ranges in GT Converted Starch Degreeof Polymerization DP 37-100 DP 25-100 DP 1-12 DP 1-24 Microfiltration38% 59% 10% 34% Retentate Microfiltration 12% 26% 40% 74% PermeateFilter Paper 33% 52% 14% 39% retentate

When the precipitated converted starch was filtered using filter paper,and the retentate was dried in the oven (50° C.). Differential scanningcalorimetry (DSC) data showed two melting peaks of 116.03° C. (13.74J/g) and 138.79° C. (0.3879 J/g) before heat-moisture treatment (FIG.6), and two melting peaks at 117.45° C. and about 140° C. with totalenthalpy of 21.23 J/g after heat-moisture treatment (FIG. 7). Theheat-moisture treatment was done at 250° F. for 1.5 hours at 25%moisture.

When the retentate was collected using microfiltration, DSC data showedtwo melting peaks of 114.9° C. and 138.79° C. with total enthalpy of19.83 J/g before heat-moisture treatment (FIG. 8), and two melting peaksat 117.07° C. and about 140° C. with total enthalpy of 21.50 J/g afterheat-moisture treatment (FIG. 9).

EXAMPLE 10

GT enzyme treatment of dent starch:

Dent Starch Pearl-C (DS 89.56%) was weighed (502.5 g), and 2497.5 gdeionized (D.I.) water and 135 mg CaCl₂.2H₂O were added to starch (15%starch slurry). The pH of starch slurry was adjusted to 5.5 using 2NNaOH solution. The starch slurry was jet cooked (285-290° F., 140-143°C.), and usually the dry solids content decreased from 15% to 13.19%.The pH was adjusted to 5.7 if it was different. 550 g of starch slurrywas weighed to each of several 1000 ml reactors. The GT enzyme was addedaccording to the quantity of dry solids in each of reactors, asexplained further below. The starch and GT enzyme mixture were incubatedin water bath at 80° C. up to 24 hr. Samples (about 5 ml) were drawn toanalyze the branch chain length.

Debranching GT converted starch:

A wet GT converted sample (about 13% dry solids) was heated with a tightcap in microwave at full power until it became a fluid. Samples (192±25mg) were weighed in 10 ml tubes, and 2.5 ml purified (HPLC grade) waterwas added. For a dry sample, 25 mg dry starch was weighed to bedissolved in 2.5 ml purified HPLC grade water. The starch wassolubilized in solution (about 1% solid) by microwave. The hot starchsolution cooled down in hot tap water (about 50° C.), and 50 μlisoamylase [10 mg/ml isoamylase (1,280,000 U/g solid) in 0.1 N NaOAcbuffer, pH 4.5] was added to the starch solution. The starch andisoamylase mixture was incubated in an oven at 55° C. for 2 hr. Thestarch and isoamylase mixture was heated to above 100° C. to inactivateisoamylase. The starch solution was cooled down using hot tap water(about 50° C.), and 0.1 g Dowex MR-3 resin was added to the starchsolution and shaken for 1 min to remove NaOAc. The starch solution wasfiltered through a 0.45 μm pore size Millipore filter attached to a 3 mlsyringe. The filtered samples were injected into the HPLC with SEC orGPC column.

Optimization of DP of chains of dent starch at differentglucanotransferase (GT) dosages over 24 hr reactions (80° C., pH 5.7):

Four different dosages of GT were tried: 1.25, 2.5, 5, and 10 ml/100 gstarch. Surprisingly, most of the changes in DP values occurred in thefirst 4 hr, and the end DP values are different at different dosages.

FIG. 10 shows the percentage DP 37-100 changes at the four dosages over24 hr. The DP 37-100 components are desirable for resistant starch, andincreased greatly in the first 4 hr of reaction. At the high dosage (10ml/100 g starch), there were decreases of DP 37-100 after 6 hr ofreaction. At the dosage of 5 ml/100 g starch, there was a decrease in DP37-100 after 22 hr of reaction.

FIG. 11 shows the percentage DP 25-100 changes at the four dosages over24 hr. The pattern of changes is exactly the same as for DP 37-100. TheDP 25-37 components may be desirable for resistant starch with lessheat-stability, and increased greatly in the first 4 hr of reaction. Atthe high dosage (10 ml/100 g starch), there were decreases of DP 25-100after 6 hr of reaction. At the dosage of 5 ml/100 g starch, there was adecrease in DP 37-60 after 22 hr of reaction.

FIGS. 12 and 13 show the percentage DP 1-24 and DP 1-12 changes at thefour dosages over 24 hr. The DP 1-24 components, especially DP 1-12, areundesirable for resistant starch, and decreased greatly in the first 4hr of reaction. At the high dosage (10 ml/100 g starch), there wereincreases of DP 1-24 after 6 hr of reaction.

FIG. 14 shows the percentage DP 100+ changes at the four dosages over 24hr. The DP 100+ components are undesirable for resistant starch, anddecreased greatly in the first 4 hr of reaction.

FIG. 15 shows the best DP peaks over 24 hr reactions at five differentGT enzyme dosages. The best DP peak over 24 hr was directly correlatedwith the GT enzyme dosage. An increase of reaction time at a lowconcentration of GT enzyme did not give a high peak DP as a highconcentration of GT enzyme did.

Effects of high dosages of glucanotransferase (GT) on DP of branchchains:

In previous studies, it was found that with increment of enzyme dosages(from 1.25 to 10 ml/100 g starch), the DP of final product increased. Inthis experiment, we attempted to find out at what enzyme dosage the DPof final product would not increase with increment of GT or the plateauof plot of DP with GT concentration. A 15% starch solution was addedwith 10, 12.5 and 15 ml GT/100 g starch and the reactions were conductedat 80° C., pH 7.5. Samples were taken at 2, 4, 6, and 22 hrs. Theresults are shown in FIGS. 16-21.

The increase of GT dosage from 10 to 15 ml/100 g of starch gave somebenefit, but the increase of percentage of DP 37-100, and the reductionsof DP 1-24 and 100+ were far less compared to those observed when enzymedosages were increased from 1.25 to 10 mV/100 g starch.

EXAMPLE 11

GT enzyme treatment of dent starch:

Dent Starch Pearl-C (DS 89.56%) was weighed (502.5 g), and 2497.5 g D.I.water and 135 mg CaCl₂.2H₂O were added to starch (15% starch slurry).The pH of the starch slurry was adjusted to 5.5 using 2N NaOH solution.The starch slurry was jet cooked (285-290° F., 140-143° C.), and usuallythe dry solids decreased from 15% to 13.19%. The pH was adjusted to 5.7if it was different. 550 g of starch slurry was weighed to each ofseveral 1000 ml reactors. The GT enzyme was added according to thequantity of dry solids in each of several reactors. The starch and GTenzyme mixture were incubated in water bath at 80° C. up to 24 hr.Samples (about 5 ml) were drawn to analyze the branch chain length.

Debranching GT converted starch:

A wet GT converted sample (about 13% solid) was heated with tight cap inmicrowave at full power until it became a fluid. Samples (192±25 mg)were weighed in 10 ml tubes, and 2.5 ml purified (HPLC grade) water wasadded. For a dry sample, 25 mg dry starch was weighed to be dissolved in2.5 ml purified HPLC grade water. The starch was solubilized in solution(about 1% solid) by microwave. The hot starch solution cooled down inhot tap water (about 50° C.), and 50 μl isoamylase [10 mg/ml isoamylase(1,280,000 U/g solid) in 0.1 N NaOAc buffer, pH 4.5] was added to thestarch solution. The starch and isoamylase mixture was incubated in anoven at 55° C. for 2 hr. The starch and isoamylase mixture was heated toabove 100° C. to inactivate isoamylase. The starch solution was cooleddown using hot tap water (about 50° C.), and 0.1 g Dowex MR-3 resin wasadded to the starch solution and shaken for 1 min to remove NaOAc. Thestarch solution was filtered through 0.45 μm pore size Millipore filterattached to a 3 ml syringe. The filtered samples were injected into theHPLC with SEC or GPC column.

Debranching GT converted starch in DMSO solution:

Debranching of GT converted starch in an experiment in which STAR-DRI 10maltodextrin was added was conducted in DMSO solution. Dry starch (35mg) was dissolved in 1 ml aqueous DMSO (DMSO:water=9:1 v/v) or wetsamples (269 mg, 13% DS in GT converted samples) are dissolved in 0.9 mlpure DMSO. The starch solution was heated in boiling water bath withstirring for 3 hr. The starch solution was then cooled to 39° C., and3.5 ml warm sodium acetate buffer (39° C., 50 mM) was added. 100 μlisoamylase [10 mg/ml isoamylase (1,280,000 U/g solid) in 0.1 N NaOAcbuffer, pH 4.5] was added to the starch solution. The starch andisoamylase mixture was incubated in a water bath at 39° C. for 2 hr. Thestarch and isoamylase mixture was heated in boiling water for 20 min,and then cooled down to 39° C. 100 μl isoamylase was added and themixture was incubated for 16 hr. After debranching, the starch solutionwas heated in boiling water bath for 20 min, and cooled down to warmtemperature. A 2 mL aliquot of the mixture is diluted with 2 mL of pureDMSO. The DMSO mixtures (about 5 mg starch/ml) were heated in a boilingwater bath for 20 min, allowed to cool to warm temperature. Dowex MR-3resin (0.5) g was added to the starch solution and shaken for 1 min toremove NaOAc. The starch solution was filtered through a 0.45 μm poresize Millipore filter attached to a 3 ml syringe. The filtered sampleswere injected into the HPLC with SEC or GPC column.

Glucanotransferase (GT) Activity at Different Reaction Temperatures (75,80, and 85° C.):

In this experiment, a 15% starch solution with 5 ml GT/100 g starch wasreacted at 75, 80 and 85° C., and samples were taken at 1, 2, 4, 6, 8,10 and 22 hrs after addition of GT. The results are shown in FIGS.22-23.

For a short reaction time (6 hr or less), GT converted starch had ahigher proportion of DP 37-100 at a high reaction temperature (85° C.).However, for a long reaction time (8 hr or longer), GT converted starchhad a higher proportion of DP 37-100 at lower temperatures (75 and 80°C.).

As shown in FIGS. 24-26, for a short reaction time (6 hr or less), GTconverted starch had a lower proportion of DP 1-24 or 1-12 at a highreaction temperature (85° C.). However, for a long reaction time (8 hror longer), GT converted starch had a lower proportion of DP 1-24 or1-12 at lower temperatures (75 and 80° C.).

As shown in FIG. 6, for a short reaction time (6 hr or less), the trendwas not clear for the DP 100+ fraction. For a long reaction time (8 hror longer), GT converted starch had a lower proportion of DP 100+ athigher temperature (80 and 85° C.). It is likely that at highertemperature, less starch retrogradation occurred and GT enzyme couldwork on the DP 100+ fraction more efficiently.

Glucanotransferase (GT) Activity at High Temperature:

In a previous reaction temperature study (75, 80 and 85° C.), thehighest percentages of DP 37-100 were similar (close to 29%) at threedifferent temperature but the highest percentages of DP 37-100 wereearly at 85° C., and later at 80 and 75° C. There was a detrimentaleffect (decrease of DP) if the reaction lasted longer than the optimum(highest peak DP or highest DP “37-100”). An experiment was performed 1)to examine the DP 37-100 and peak DP in the early stage (0.5 hr) athigher temperature, and 2) to test the GT heat stability by pre-heatingthe GT enzyme at 85° C. for 4 hr.

In this experiment, GT 10 ml/100 g starch was used in all fourtreatments. GT reactions with starch were conducted at 80° C., 85° C.,85° C. with pre-heat converted GT (85° C. for 4 hr), and 90° C. FIG. 28shows the results. In the first 1.5 hr, the percentage of DP 37-100 washigher at 95° C. However, pre-heating GT at 85° C. gave a higher DP37-100 than GT reaction at 85° C. The DP 25-100 fraction (FIG. 29)followed a similar trend.

The results regarding the short DP chains (DP 1-12 and 1-24) wereinconclusive because of the variation of the data but no significantdetrimental effect was seen at higher temperatures (FIGS. 30 and 31).The peak DP (FIG. 30) showed that reaction at 90° C. gave a higher peakDP than reaction at 80° C. within 1.5 hr, and preheating of GT at 85° C.for 4 hr gave a higher peak DP than reaction at 85° C. withoutpreheating GT.

FIG. 32 shows that DP 100+ was lower at 90° C. FIG. 33 shows that thepeak DP was higher at 90° C. on the first 1.5 hr, although the trend wasnot very clear.

The peak DP for reaction at longer times (from 2 hr to 8 hr) is shown inFIG. 34. Over a 2 hr reaction, lower temperature reactions seem to bebetter than higher temperature reactions for peak DP.

Effect of Addition of STAR-DRI 10 on the GT Converted Starch:

Dent Starch Pearl-C (15%) was jet-cooked (285-290° F.), and pH wasadjusted to 5.7. STAR-DRI 10 maltodextrin was dissolved in DI water in1:2 ratio, solubilized at 80° C., and adjusted to pH 5.7. The starchslurry was incubated at 80° C. and four tests were conducted: 1. starchslurry+10 ml GT/100 g starch; 2. starch slurry+10 ml GT/100 g starch+25%STAR-DRI 10 based on dry starch; 3. starch slurry+10 ml GT/100 g starchand react for 2 hr, then 25% STAR-DRI 10; 4. starch slurry+12.5 mlGT/100 g starch+25% STAR-DRI 10. Samples were drawn at 2, 4, and 6 hr.The samples were debranched. The results are shown in FIGS. 35-38.

The addition of STAR-DRI 10 decreased the DP in GT converted dentstarch. It decreased DP overall, whether STAR-DRI 10 was added at thebeginning or after 2 hr of reaction. It even decreased the overall DP ifa comparable amount of GT enzyme was added to compensate the increase ofthe starch solids in the solution.

EXAMPLE 12

GT enzyme treatment of dent starch:

Dent Starch Pearl-C (DS 89.56%) was weighed (502.5 g), and 2497.5 g D.I.water and 135 mg CaCl₂.2H₂O were added to starch (15% starch slurry).The pH of the starch slurry was adjusted to 5.5 using 2N NaOH solution.The starch slurry was jet cooked (285-290° F. 140-143° C.), and usuallythe dry solids decreased from 15% to 13.19%. The pH was adjusted to 5.7if it was different. 550 g of starch slurry was weighed to each ofseveral 1000 ml reactors. The GT enzyme was added according to thequantity of dry solids in each of the reactors. The starch and GT enzymemixture were incubated in water bath at 80° C. up to 24 hr. Samples(about 5 ml) were drawn to analyze the branch chain length.

Debranching GT converted starch:

A wet GT converted sample (about 13% solid) was heated in a test tubewith a tight cap in a microwave oven at full power until it became afluid. Samples (192±25 mg) were weighed in 10 ml tubes, and 2.5 mlpurified (HPLC grade) water was added. For a dry sample, 25 mg drystarch was weighed to be dissolved in 2.5 ml purified HPLC grade water.The starch was solubilized in solution (about 1% solid) by microwave.The hot starch solution cooled down in hot tap water (about 50° C.), and50 μl isoamylase [10 mg/ml isoamylase (1,280,000 U/g solid) in 0.1 NNaOAc buffer, pH 4.5] was added to the starch solution. The starch andisoamylase mixture was incubated in an oven at 55° C. for 2 hr. Thestarch and isoamylase mixture was heated to above 100° C. to inactivateisoamylase. The starch solution was cooled down using hot tap water(about 50° C.), and 0.1 g Dowex MR-3 resin was added to the starchsolution and shaken for 1 min to remove NaOAc. The starch solution wasfiltered through 0.45 μm pore size Millipore filter attached to a 3 mlsyringe. The filtered samples were injected into the HPLC with SEC orGPC column.

From the previous experiments, the best DP peak over 24 hr was directlycorrelated with GT enzyme dosage. An increase of reaction time at lowconcentrations of GT enzyme did not give a high peak DP as highconcentrations of GT enzyme did. It is hypothesized that either the GTenzyme is inactivated after first four hr of reaction or starch isretrograded so that GT can not effectively work on the starch.

To determine which factor was slowing down the reaction, threeexperiments were performed: 1. GT was added right after jet cooking (0hr) at full dosage (7.5 ml/100 g starch); 2. GT was added right afterjet cooking in ⅓ of the total dosage (2.5 ml/100 g starch at 0 hr), thesecond ⅓ dosage (2.5 ml/100 g starch) after 2.5 hr reaction, and thethird ⅓ dosage (2.5 ml/100 g starch) after 4 hr reaction; 3. GT wasadded after starch was incubated at 80° C. for 5.5 hr after jet cooking.

The experiment was intended to reveal the stability of the GT enzyme at80° C. and the effect of retrogradation of starch on enzyme activity. Ifstarch retrogradation has no effect on the enzyme activity and theenzyme is stable, then the end product (debranched starch GPC profiles)would be the same after extended enzyme reaction. Also, with each GTenzyme addition (2.5 ml/100 g starch, three times), the debranchedstarch GPC profiles would change until they reach the same profile asone full dosage (7.5 ml/100 g starch). The results are shown in FIGS.39-40.

FIG. 39 shows that the percentage of DP 37-100 was the same with thesame GT dosage (7.5 ml/100 g starch) after 4 hr reaction regardless ofwhether the GT was added right after jet cooking (0 hr) or after thestarch was incubated at 80° C. for 5.5 hr. The same trend was true forDP 25-100. The DP 25-37 included may be desirable for resistant starchbut with less heat-stability.

When ⅓ of the total dosage of GT was added right after jet cooking (2.5ml/100 g starch at 0 hr), DP 37-100 increased in the initial 1.5 hr andthen decreased from 1.5 hr to 2.5 hr. With the second addition of GT(2.5 ml/100 g starch), the DP 37-100 increased quickly. When the thirddosage GT (2.5 ml/100 g starch) was added, the change was not as greatas with the addition of the second dosage. It is hypothesized thateither some retrogradation occurred after reaction with addition of thesecond dosage GT or the reaction was close to the equilibrium after thereaction with the second GT dosage. More DP 37-100 material was obtainedwhen GT was added at ⅓ dosage a time than when it was added in a singledosage.

FIGS. 41 and 42 show that the percentage of DP 1-24 and 1-12 were alittle higher when GT was added right after jet cooking (0 hr) thanafter the starch was incubated at 80° C. for 5.5 hr. However, thedifference was less than 2%.

When GT was added right after jet cooking in ⅓ of dosage (2.5 ml/100 gstarch at 0 hr), DP 1-24 decreased from 0 to 1 hr but increased from 1to 3 hr, decreased sharply with addition of the second dosage of GT, andthen continued to decrease with the third dosage of GT. There was lessDP 1-24 when GT was added at ⅓ dosage at a time instead of one fulldosage.

FIG. 43 shows that DP 100+ dropped quickly and was close to the endvalue after initial 2 hr enzyme reaction. It is surprising that eventhough low dosage GT (2.5 ml/100 g starch) was added DP 100+ dropped toa similar end value as with high dosage (7.5 ml/100 g starch) after 2 hrreaction. It was also unexpected that DP 100+ increased from about 0.5%to 4% with the second addition of GT (2.5 ml/100 g starch).

FIG. 43 also shows that incubation of the starch slurry at 80° C. for5.5 hr after jet cooking gave a higher end value of DP 100+. It islogical to conclude that some amylose retrograded at 80° C. for 5.5, andthat GT can not work on these retrograded amyloses.

More data (every 0.5 hr) were obtained from the reaction when GT wasadded in ⅓ of the dosage a time. The detailed DP changes in 8 hrreaction are shown in FIG. 44.

The best peak DPs of GT enzyme (7.5 ml/100 g starch) converted starchwere the same with the same GT dosage (7.5 ml/100 g starch) after 6 hrreaction, regardless of whether the GT was added right after jet cooking(0 hr) or after the starch was incubated at 80° C. for 5.5 hr. (See FIG.45.) When GT was added in ⅓ of the total dosage a time (2.5 ml/100 gstarch at 0, 2.5, and 4 hr respectively), the best peak DP increasedprogressively. The final best peak DP, after the whole dosage (7.5ml/100 g starch) was added, was better than addition of the whole dosagein one time.

EXAMPLE 13

Resistant starch prepared according to the present invention was used toreplace 51.7% of the flour in a cookie bake test (American Associationof Cereal Chemists (AACC) test 53-10). The resistant starch had beenpassed through a US mesh 40 sieve and was collected on a US mesh 200sieve, with the fines passing through the 200 mesh sieve. The particlesize mean was 202.5 μm and the mode was 185.4 μm.

As analyzed by test AACC 56-11, the starch sample was found to be higherin water holding capacity than pastry flour, however, this measurementdoes not account for what happens to the ingredients during the heatingcycle of a cookie during baking.

TABLE 4 Water holding (AACC 56-11, sodium carbonate solvent only)Ingredient % AWRC Pastry flour 64.61 (0.91 g solvent/g flour) Starchsample 99.01 (1.31 g solvent/g starch)

An Instron tester was used to measure dough firmness and stickiness.(Instron Corp., Canton, Mass.; ½ inch ball probe; trigger force=10 g;pretest speed=5 mm/s; test speed=2 mm/s; post test speed=10 mm/s;distance of penetration=15 mm.) 150 grams of dough were weighed into apan which had a height of 8.4 cm, a width of 3.2 cm, and a length of10.2 cm. The dough was compressed into the pan with a single stroke of arolling pin. Average values of at least three Instron compressions wererecorded. If a resistant starch imbibes excessive water, the doughbecomes firm. The starch sample used in this experiment produced a doughthat was less firm (lower maximum load) than the pastry flour and wasfound to be less sticky as measured by force to release the probe aftercompression (min. force).

TABLE 5 Cookie Dough Performance as measured by Instron Cookie Max load(g) Min force (g) All flour control 272.53 −194.63 51.7% replacement offlour 178.29 −120.19 with resistant starch

According to AOAC (Association of Official Analytical Chemists) method991.43, 71.94% fiber was present in the resistant starch ingredientprior to baking, and 88.3% of that material was calculated as fiberfollowing cookie baking.

TABLE 6 Cookie Cookie formula formula Complete Complete moisture/fatmoisture/fat cookie formula cookie formula free free Ingredient Control% Test % Control % Test % Nonfat dry milk 0.47 0.47 0.73 0.73 Salt 0.580.58 0.90 0.90 Soda 0.47 0.47 0.73 0.73 Fine granulated sugar 19.6719.67 30.43 30.43 Fat 18.73 18.73 — — High fructose corn 0.50 0.50 0.770.77 syrup (42% fructose) ds Ammonium 0.24 0.24 0.37 0.37 bicarbonateFlour (pastry flour) ds 42.72 20.60 66.09 31.87 Resistant starch ds —22.11 — 34.20 water 16.63 16.63 — —

TABLE 7 Control Test Cookie % TDF 3.54 23.44 (fat and moisture free)Contribution to TDF before 0 0 baking from flour Contribution to TDFbefore 0 24.60 baking from resistant starch (TDF = 71.94%) Contributionto TDF after baking 3.54 1.71 from flour Contribution to TDF afterbaking 0 21.73 from resistant starch Loss of resistant starch TDF % —11.67 during baking

The cookie height for the control pastry flour cookie was greater thanthe height of the cookie that contained resistant starch. Additionally,cookie spread (width) was less for the control and greater for theresistant starch-containing cookie. Greater spread and reduced height isdue to the low water holding property of the resistant starch andindicates that the resistant starch did not hydrate or partiallygelatinize during the baking process, but remained relatively unchanged.

TABLE 8 Cookie Performance Width (average of 4 Cookie cookies) HeightAll flour control  8.0 cm 1.1 cm 51.7% replacement of flour 8.85 cm 0.9cm with resistant starch

The preceding description of specific embodiments of the invention isnot intended to be a list of every possible embodiment of the invention.Persons skilled in the art will recognize that other embodiments wouldbe within the scope of the following claims.

1. A process for producing a starch, comprising: treating a feed starchthat comprises amylopectin with glucanotransferase to produce achain-extended starch; and treating the chain-extended starch with adebranching enzyme to produce a starch product that comprises amylosefragments; wherein at least about 38% by weight of the amylose fragmentshave a degree of polymerization (DP) of at least about
 35. 2. Theprocess of claim 1, further comprising recovering the amylose fragments.3. The process of claim 1, wherein the feed starch is from dent corn,waxy corn, potato, tapioca, rice, pea, wheat, waxy wheat, or acombination of two or more thereof.
 4. The process of claim 1, whereinthe feed starch is treated with glucanotransferase in aqueous solutionor suspension at a temperature of about 70-100° C. and a pH of about5.0-6.0.
 5. The process of claim 1, wherein the debranching enzyme isisoamylase or pullulanase.
 6. The process of claim 1, wherein the starchproduct has a polydispersity of about 2-4.
 7. The process of claim 1,further comprising membrane filtering a solution or dispersion of thestarch product to increase the concentration of amylose fragments thathave a degree of polymerization (DP) of at least about
 35. 8. Theprocess of claim 1, wherein at least about 40% by weight of the amylosefragments have a degree of polymerization (DP) of at least about
 35. 9.The process of claim 7, wherein after membrane filtration at least about50% by weight of the amylose fragments have a degree of polymerization(DP) of at least about
 35. 10. The process of claim 1, wherein thestarch product has a peak melting temperature of greater than about 105°C.
 11. The process of claim 1, further comprising heating the starchproduct in aqueous solution or suspension at a temperature of at leastabout 90° C.
 12. The process of claim 11, wherein the starch product isheated in aqueous solution or suspension at a temperature of at leastabout 98° C.
 13. The process of claim 1, wherein the starch product isthermally stable in water at temperatures up to at least about 90° C.14. The process of claim 13, wherein the starch product is thermallystable in water at temperatures up to at least about 100° C.
 15. Theprocess of claim 1, wherein the feed starch is a waxy starch, andwherein the process further comprises treating the feed starch with adebranching enzyme before the feed starch is treated withglucanotransferase.
 16. The process of claim 1, wherein theglucanotransferase is used in a dosage of about 1-15 ml per 100 grams offeed starch.
 17. The process of claim 16, wherein the glucanotransferaseis used in a dosage of about 5-12 ml per 100 grams of feed starch. 18.The process of claim 16, wherein the glucanotransferase is used in aplurality of dosages that are supplied to the feed starch at separatetimes.
 19. The process of claim 1, wherein the feed starch is treatedwith glucanotransferase for a time less than about 8 hours.
 20. Theprocess of claim 19, wherein the feed starch is treated withglucanotransferase for a time less than about 6 hours.
 21. The processof claim 1, wherein the debranching enzyme is used in a dosage of about1-10 mg per gram of starch.
 22. The process of claim 21, wherein thedebranching enzyme is used in a dosage of about 1-5 mg per gram ofstarch.